Calreticulin-mediated upregulation of anti-apoptotic Bcl-XL and Mcl-1 promotes survival of fibroblast-like synoviocytes in rheumatoid arthritis / 中华风湿病学杂志

ABSTRACT

Objective Fibroblast-like synoviocytes (FLS) play important roles in the pathogenesis of rheumatoid arthritis (RA).The present study was undertaken to investigate the mechanism of calreticulin (CRT) to promote FLS survival in RA.Methods FLS were isolated by enzymatic digestion of synovial tissue specimens obtained from RA and osteoarthritis (OA) patients and cultured in vitro.The expression of Bcl-XL and Mcl-1 in FLS at mRNA and protein level was detected by quantitative-polymerase chain reaction (q-PCR),Western blotting and immunofluorescence respectively.RA and OA FLS were cultured with different concentrations of recombinant human CRT for 48-72 h,the expression of Bcl-XL and Mcl-1 was detected by q-PCR and Western blotting.The proliferation of RA FLS following CRT stimulation was determined by MTT assay.Results ① Compared with FLS from OA patients (1.00±0.39;1.00±0.46),the anti-apoptotic Bcl-XL and Mcl-l mRNA expression (14.51 ±2.20;12.82±1.80) was significantly higher in the FLS from RA patients (t=10.47,1 1.02;P＜0.01);Western blotting analysis also showed increased protein levels of Bcl-XL and Mcl-1 in RA FLS;Immunofluorescence results showed higher expression of Bcl-XL and Mcl-1 in RA at the single FLS level;② CRT up-regulated the expression of Bcl-XL and Mcl-1 in RA FLScompared with the control group (0 ng/ml),CRT stimulation at 10 ng/ml and 50 ng/ml increased the levels of Bcl-XL mRNA (1.70±0.28 vs 1.00±0.20,q=4.58,P＜0.05;1.87±0.35 vs 1.00±0.20,q=5.69,P＜O.05) and Mcl-1 mRNA (1.85±0.36 vs 1.00±0.20,q=5.63,P＜0.05;1.72±0.26 vs 1.00±0.20,q=4.77,P＜0.05) in RA FLS,while no significant effects of CRT on Bcl-XL and Mcl-1 mRNA expression were observed in OA FLS (F=1.49,1.60;P＞0.05);Western blotting results showed elevated protein levels of both Bcl-XL and Mcl-1 in RA FLS after CRT treatment at a concentration dependent manner.However,neither Bcl-XL nor Mcl-1 expression was significantly changed in OA FLS.③ MTT assay showed that CRT had no significant effect on the proliferation of RA FLS (F=2.88,P＞ 0.05).Conclusion Our results indicate that CRT-mediated up-regulation of anti-apoptotic Bcl-XL and Mcl-1 may inhibit apoptosis and promote the survival of FLS from RA patients.